血浆来源的细胞外囊泡 (pEV) 是患病生物标志物蛋白的潜在来源。然而，由于 pEV 蛋白质组丰度相对较低且富集困难，表征 pEV 蛋白质组具有挑战性。本研究提出了一种简化的工作流程，可使用最少的样本输入从癌症患者血浆中识别 EV 蛋白。从 400 μL 血浆开始，我们使用尺寸排阻色谱 (SEC) 结合基于 HiRIEF 预分级的质谱 (MS) 生成了全面的 pEV 蛋白质组。首先，我们使用对照 pEV 比较了 HiRIEF 和长梯度 MS 工作流程的性能，并使用 HiRIEF 定量了 2076 种蛋白质。在一项概念验证研究中，我们将 SEC-HiRIEF-MS 应用到一小群 (12) 转移性肺腺癌 (LUAD) 和恶性黑色素瘤 (MM) 患者中。我们还分析了同一患者的血浆样本，以研究血浆和 pEV 蛋白质组之间的关系。我们在所有样本中鉴定并定量了癌症 pEV 中的 1583 种蛋白质和血浆中的 1468 种蛋白质。虽然存在大量重叠，但 pEV 蛋白质组包括几种独特的 EV 标记和癌症相关蛋白。差异分析显示 LUAD 与 MM 组相比有 30 个 DEP，凸显了 pEV 作为生物标志物的潜力。这项工作展示了基于预分级的 MS 在全面的 pEV 蛋白质组学和 EV 生物标志物发现中的实用性。数据可通过 ProteomeXchange 获得，标识符为 PXD039338 和 PXD038528。

Plasma-derived extracellular vesicles (pEVs) are a potential source of diseased biomarker proteins. However, characterizing the pEV proteome is challenging due to its relatively low abundance and difficulties in enrichment. This study presents a streamlined workflow to identify EV proteins from cancer patient plasma using minimal sample input. Starting with 400 μL of plasma, we generated a comprehensive pEV proteome using size exclusion chromatography (SEC) combined with HiRIEF prefractionation-based mass spectrometry (MS). First, we compared the performance of HiRIEF and long gradient MS workflows using control pEVs, quantifying 2076 proteins with HiRIEF. In a proof-of-concept study, we applied SEC-HiRIEF-MS to a small cohort (12) of metastatic lung adenocarcinoma (LUAD) and malignant melanoma (MM) patients. We also analyzed plasma samples from the same patients to study the relationship between plasma and pEV proteomes. We identified and quantified 1583 proteins in cancer pEVs and 1468 proteins in plasma across all samples. While there was substantial overlap, the pEV proteome included several unique EV markers and cancer-related proteins. Differential analysis revealed 30 DEPs in LUAD vs the MM group, highlighting the potential of pEVs as biomarkers. This work demonstrates the utility of a prefractionation-based MS for comprehensive pEV proteomics and EV biomarker discovery. Data are available via ProteomeXchange with the identifiers PXD039338 and PXD038528.